Plastic film mulching (PF) can change soil properties and microbial community structure. However, the effect of PF on soil microbial interactions in different aggregate fractions is still unclear. We conducted a three-year field trial in black soil of northeastern China to evaluate the variation in soil microbial interactions and community structure in different aggregate fractions after PF. Compared with the control (CK), the PF treatment significantly decreased the humification index by 13.9% and chromophoric dissolved organic matter by 36.6% and significantly increased the fractal dimension by 4.8% and electrical conductivity by 22.8%. In addition, the PF treatment significantly decreased the relative abundances of Chloroflexi, Planctomycetes and Verrucomicrobia but significantly increased the relative abundance (RA) of Proteobacteria. Furthermore, the PF treatment significantly decreased the RA of Glomeromycota in megaaggregates (>2 mm, ME) and macroaggregates (0.25–2 mm) and the RA of Actinobacteria in microaggregates (<0.25 mm, MI), related to decreases in aggregate stability. Compared with the CK treatment, the PF treatment significantly decreased the bacterial Shannon index in ME but significantly increased the fungal Shannon index in MI. Moreover, β-1,4-glucosidase activity significantly influenced the community structure of soil bacteria and fungi. The PF treatment decreased soil microbial interactions, especially in ME. The keystone of the microbial network shifted from 0319-6A21 (Nitrospirae) to Gsoil-1167 (Actinobacteria) after PF. In short, PF decreased soil aggregate stability and microbial interactions in black soil in a subhumid area.